The present invention relates to an isolated microorganism belonging to the Mycobacterium genus, characterised in that it comprises the inactivation of the Rv0757 gene that confers a PhoP− phenotype and the inactivation of a second gene that prevents DIM production (DIM− phenotype). Additionally, the present invention comprises the use of said microorganism for the preparation of a vaccine for the immunization or prevention of tuberculosis.
The use of vaccines to prevent tuberculosis in humans has proved to be a tremendous challenge for almost a century now. BCG, derived from M. bovis, is currently the only tuberculosis vaccine in use and is the most widely used vaccine in the world. The development and generalised administration of the BCG vaccine since the beginning of the 1920s represented a significant advance, with the prospect of being able to eradicate tuberculosis from the world. However, these initial promises were not achieved and, from the results of a large number of efficacy trials, it is clear that the BCG vaccine in its current form is of limited use in controlling the disease, particularly in respiratory forms in adults in third world areas where the disease is endemic. Fine, P.E. Variation in protection by BCG: implications of and for heterologous immunity. Lancet 1995, 346(8986), 1339-1345. With more knowledge of the virulence of M. tuberculosis and immune response models that lead to the generation of protective immunity, it is possible to develop better vaccines than BCG. The observation that higher protection levels are achieved when the host is vaccinated with BCG suggests that viability and persistence are fundamental properties required for the success of a tuberculosis vaccine. In the present invention, we use a M. tuberculosis strain with the inactivated Rv0757 (phoP) gene and a second independent mutation of phoP, which prevents DIM synthesis, as a prototype single dose live vaccine, and we show that, as well as being more attenuated than BCG in immunocompromised SCID mice, it provided protection levels comparable to those conferred by BCG in mice and higher protection than BCG in guinea pigs.
The phoP gene, together with phoR, forms part of a two-component system that shows a high degree of similarity to other two-component systems that control the transcription of key virulence genes in intracellular pathogens. It also controls the expression of many other genes that are not directly involved in virulence. Groisman, E. A. The pleiotropic two-component regulatory system PhoP−PhoQ. J Bacteriol 2001, 183(6), 1835-1842. The elimination of virulence genes does not seem to be, per se, the only method for the attenuation of M. tuberculosis. It was shown that a pantothenate auxotrophic mutant of M. tuberculosis, which is incapable of de novo synthesis of pantothenic acid, persisted in SCID mice, without managing to cause the disease. Sambandamurthy, V. K., Wang, X., Chen, B. et al. A pantothenate auxotroph of Mycobacterium tuberculosis is highly attenuated and protects mice against tuberculosis. Nat Med 2002, 8(10), 1171-1174. Individual leucine auxotrophs are also strongly attenuated and incapable of replication in vivo in SCID mice. Hondalus, M. K., Bardarov, S., Russell, R., Chan, J., Jacobs, W. R., Jr. & Bloom, B. R. Attenuation of and protection induced by a leucine auxotroph of Mycobacterium tuberculosis. Infect Immun 2000, 68(5), 2888-2898. Therefore, the principle that vaccine strains based on M. tuberculosis can be successfully attenuated whilst retaining genes that are suppressed in M. bovis BCG is now generally accepted.
In the past, research into more effective vaccines than BCG was based on the notion that loss of virulence with BCG was in itself a factor that contributed to its lack of complete protective efficacy. Behr, M. A., Wilson, M. A., Gill, W. P. et al. Comparative genomics of BCG vaccines by whole-genome DNA microarray. Science 1999, 284(5419), 1520-1523. It was therefore reasoned that new attenuated mutants of M. tuberculosis, with less virulence, could be more effective as vaccines. However, a recent study has shown that natural infection with M. tuberculosis and vaccination with BCG do not differ in their capacity to bring about protective immunity against tuberculosis. Sampson, S. L., Dascher, C. C., Sambandamurthy, V. K. et al. Protection elicited by a double leucine and pantothenate auxotroph of Mycobacterium tuberculosis in guinea pigs. Infect Immun 2004, 72(5), 3031-3037. This raises questions as to whether or not it is possible to improve BCG by rational attenuation of M. tuberculosis. Within this context, the observation that the mutant M. tuberculosis strain of the present invention with the combination of 2 independent mutations 1.—in synthesis of the PhoP protein and 2.—in DIM synthesis is more attenuated than BCG in the SCID mouse model, even when applied at a dose 10 times higher than those of BCG, and the greater degree of protection than BCG in the guinea pig model is particularly surprising and significant.